Purchasing a Stopped-flow Spectrometer for Characterization of Reaction Intermediates of Hydrogenase Enzyme Models Stopped-flow spectroscopy has become a major tool in the characterization of reaction intermediates of synthetic models of the active sites of hydrogenases in this project. This NIH- supported work focuses on both broad classes of hydrogenases ([FeFe]- and [NiFe]) which are relevant to the metabolism of pathogens and significant in energy technology relative to hydrogen production. The goal of the research is to elucidate mechanisms by which these enzymes operate as well as the pathways by which they are biosynthesized. The PI's group and collaborators have shown that the binuclear cluster of [FeFe] hydrogenase is formed with sulfur donated by cysteine of an [Fe(Cys)(CO)2(CN)] organometallic precursor. This work sets the stage for further investigation of the H-cluster bioassembly. Very recent work using electron paramagnetic resonance in conjunction with freeze-quench has identified three intermediates that connect [Fe(cysteine)(CO)2(CN)] to the H-cluster. We anticipate generating downstream intermediates in the biosynthesis and evaluating their conversions. We however need tools for monitoring rates. The proposed stopped-flow spectrometer ideally meets this need. We have previously been using a stopped-flow spectrometer from Professor Robert B. Gennis group in the Department of Biochemistry on the same floor of the building with the PI group. This spectrometer, Applied Photophysics Ltd. SX18.MV, was purchased > 20 years ago and has been used frequently ever since. The expected instrument lifetime, according to Applied Photophysics representatives is <15 years, and ours has been heavily used for more than 20 years. Our instrument has been unreliable for the past few years, and because the instrument is so old and no longer supported by the company, repairs have been costly, both in parts/labor and in long delays (often weeks) before the stopped-flow spectrometer can be used for projects. In addition, the current spectrometer does not have a low temperature control unit. Since most of reaction intermediates in our hydrogenase models are not stable at room temperature, we need to add such a unit to the spectrometer. Since kinetic measurement is becoming a main technique that we use for this NIH project, the unreliability of the stopped-flow spectrometer and costly repairs have become a huge bottleneck in our projects. Therefore, we are seeking supplemental funding to replace our outdated and aging instrument with a modern model (AppliedPhotophysics SX20) and to add a new capability of low temperature control unit in order to increase productivity.